The present invention relates to novel compounds that are useful for inhibition, alteration, or prevention of cell adhesion and cell adhesion-mediated pathologies. This invention also relates to pharmaceutical formulations comprising these compounds, and methods of using them for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. The compounds and pharmaceutical compositions of this invention can be used as therapeutic or prophylactic agents. They are particularly well suited for the treatment of many inflammatory and autoimmune diseases.
Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. As such, cell adhesion constitutes one of the fundamental mechanisms underlying numerous biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hematopoietic cells out of blood vessels and to the site of injury. As such, cell adhesion plays a role in numerous pathologies such as, for example, inflammation and immune reactions in mammals.
Investigations into the molecular basis for cell adhesion have revealed that various cell-surface macromoleculesxe2x80x94collectively known as cell adhesion molecules or receptorsxe2x80x94mediate cell-cell and cell-matrix interactions. For example, proteins of the superfamily called xe2x80x9cintegrinsxe2x80x9d are key mediators in adhesive interactions between hematopoietic cells and their microenvironment (M. E. Hemler, xe2x80x9cVLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.xe2x80x9d Ann. Rev. Immunol., 8, p. 365 (1990)). Integrins are non-covalent heterodimeric complexes consisting of two subunits called xcex1 and xcex2. There are at least 17 different xcex1 subunits (xcex11-xcex110, xcex1-L, xcex1-M, xcex1-D, xcex1-X, xcex1-IIB, xcex1-V and xcex1-E) and at least 9 different xcex2(xcex21-xcex29) subunits which have been identified to date. Based on the type of its xcex1 and xcex2 subunit components, each integrin molecule can be categorized into a subfamily.
Integrin xcex14xcex21, also known as very late antigen-4 (xe2x80x9cVLA-4xe2x80x9d) or CD49d/CD29, is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell-matrix adhesive interactions (M. E. Hemler, Ann. Rev. Immunol., 8, p. 365 (1990)). It serves as a receptor for the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-1 (xe2x80x9cVCAM-1xe2x80x9d), as well as for the extracellular matrix protein fibronectin (xe2x80x9cFNxe2x80x9d) (Ruegg et al., J. Cell Biol., 177, p. 179 (1991); Wayner et al., J. Cell Biol., 105, p. 1873 (1987); Kramer et al., J. Biol. Chem., 264, p. 4684 (1989); Gehlsen et al. Science, 24, p. 1228 (1988)). Anti-VLA-4 monoclonal antibodies (xe2x80x9cmAb""sxe2x80x9d) have been shown to inhibit VLA-4-dependent adhesive interactions both in vitro and in vivo (Ferguson et al. Proc. Natl. Acad. Sci., 88, p. 8072 (1991); Ferguson et al., J. Immunol., 150, p. 1172 (1993)). Results of in vivo experiments suggest that the inhibition of VLA-4-dependent cell adhesion may prevent, inhibit or alter several inflammatory and autoimmune pathologies. (R. L. Lobb et al., xe2x80x9cThe Pathophysiologic Role of xcex14 Integrins In Vivoxe2x80x9d, J. Clin. Invest., 94, pp. 1722-28 (1994)).
In order to identify the minimum active amino acid sequence necessary to bind VLA-4, Komoriya et al. synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin. (xe2x80x9cThe Minimal Essential Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within the Alternatively Spliced Type III Connecting Segment Domain of Fibronectin Is Leucine-Aspartic Acid-Valinexe2x80x9d, J. Biol. Chem., 266 (23), pp. 15075-79 (1991)). They identified an 8-amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val and Leu-Asp-Val-Pro-Ser, that possessed inhibitory activity against FN-dependent cell adhesion. These results suggested that the tripeptide Leu-Asp-Val was the minimum sequence for cell-adhesion activity. It was later shown that Leu-Asp-Val binds only to lymphocytes that express an activated form of VLA-4, thus casting doubt on the utility of such a peptide in vivo (E. A. Wayner et al., xe2x80x9cActivation-Dependent Recognition by Hematopoietic Cells of the LDV Sequence in the V Region of Fibronectinxe2x80x9d, J. Cell. Biol., 116(2), pp. 489-497 (1992)). However, certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo (T. A. Ferguson et al., xe2x80x9cTwo Integrin Binding Peptides Abrogate T-cell-Mediated Immune Responses In Vivoxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991); and S. M. Wahl et al., xe2x80x9cSynthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitmentxe2x80x9d, J. Clin. Invest., 94, pp. 655-62 (1994)). A cyclic pentapeptide which can inhibit both VLA-4 and VLA-5 adhesion to FN has also been described. (See, e.g., D. M. Nowlin et al. xe2x80x9cA Novel Cyclic Pentapeptide Inhibits xcex14xcex21 and xcex15xcex21 Integrin-mediated Cell Adhesionxe2x80x9d, J. Biol. Chem., 268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862). This pentapeptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a common motif in the recognition site for several extracellular-matrix proteins.
Examples of other VLA-4 inhibitors have been reported, for example, in copending United States patent application 08/376,372, specifically incorporated by reference herein. U.S. Ser. No. 376,372 describes linear peptidyl compounds containing xcex2-amino acids which have cell adhesion inhibitory activity. International patent applications WO 94/15958 and WO 92/00995, specifically incorporated by reference, describe cyclic peptide and peptidomimetic compounds with cell adhesion modulating activity. International patent applications WO 93/08823 and WO 92/08464 (specifically incorporated by reference herein) describe guanidinyl-, urea- and thiourea-containing cell adhesion modulating compounds. U.S. Pat. No. 5,260,277 describes guanidinyl cell adhesion modulation compounds, and is also specifically incorporated herein.
Despite these advances, there remains a need for low molecular weight, specific inhibitors of VLA-4 dependent cell adhesion that have improved pharmacokinetic and pharmacodynamic properties such as oral bioavailability and significant duration of action. Such compounds would provide useful agents for treatment, alteration, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 binding.
The compounds of the present invention are inhibitors of the VLA-4 integrin, thereby blocking the binding of VLA-4 to its various ligands, such as VCAM-1 and regions of fibronectin. Thus these compounds are useful in inhibiting cell adhesion processes including cell activation, migration, proliferation and differentiation. These compounds are useful for inhibition, prevention and suppression of VLA-4-mediated cell adhesion and pathologies associated with that adhesion, such as inflammation and immune reactions, including for example, multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, septic arthritis, type 1 diabetes, organ transplantation, restenosis, autologous bone marrow transplantation, inflammatory sequelae of viral infections, myocarditis, inflammatory bowel disease including ulcerative colitis and Crohn""s disease, certain types of toxic and immune-based nephritis, contact dermal hypersensitivity, psoriasis, tumor metastasis, multiple myeloma, and atherosclerosis. The compounds of this invention may be used alone or in combination with other therapeutic or prophylactic agents to inhibit, alter, prevent or suppress cell adhesion. This invention also provides pharmaceutical formulations containing these VLA-4-mediated cell adhesion inhibitors and methods of using the compounds and compositions of the invention for inhibition of cell adhesion.